Quantifying Cardiac Oxidative Stress

Cardiac level of ROS was determined by using two independent methods. As we previously described 21 (link), O₂^- level in myocardium was detected with dihydroethidium (DHE) fluorescent stain of LV frozen sections (5 μm) by incubation of DHE (5 µM for 1 h at 37 °C). Images were digitized (×400 magnification, 6 fields per heart) under a fluorescence microscope (DP72; Olympus, Tokyo, Japan) with excitation/emission at 488/610 nm. In addition, chloromethyl derivative CM-H2DCFDA (DCF) fluorescent staining of isolated adult mouse cardiomyocytes was used to test ROS level in the myocardium. Cardiomyocytes isolated through Langendorff perfusion system were incubated with 5 µM DCF for 30 min at 37 ℃. Images were obtained using a Leica TCS SP8 STED 3X confocal microscope with 40× 1.3 NA oil immersion objective (excitation 488 nm, emission 525 nm) with fixed scanning parameters. An average of 50 cells of each heart were analyzed for the intensity of DCF fluorescence in ImageJ software.